This research proposal focuses on elucidating heart developmental genetics in the primitive chordate Ciona intestinalis. Initial efforts will focus on the regulation and function of the single Ciona ortholog to the vertebrate Nkx 2.x gene family, Ci-Nkx. The ability to test reporter constructs through electroporation will allow rapid identification of the regulatory elements which control Ci-Nkx expression. This technique will also permit prompt identification of functionally important binding sites within these elements. Identification of functional binding sites will be employed to identify factors which regulate Nkx. Misexpression and repression of Ci-Nkx will be employed to determine its functional significance and to identify downstream target genes. Research on Nkx 2.5 in vertebrate heart development has been hindered by the presence of numerous paralogs. Therefore, analysis of Ciona Nkx regulation and function can make a significant contribution towards understanding conserved aspects of vertebrate heart genetics. A fundamental understanding of heart genetics is essential to the development of medical treatments for heart-related diseases and birth defects.